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Preface

This risk-benefit analysis is part of Forever Healthy's "Rejuvenation Now" initiative that seeks to continuously identify potential rejuvenation therapies and systematically evaluate their risks, benefits, and associated therapeutic protocols to create transparency.

Section 1: Overview 

Motivation

NAD+ is a pyridine nucleotide found in all living cells. It plays an important role in energy metabolism and is a substrate for several enzymes (including those involved in DNA repair). NAD+ levels may decline markedly with age (Massudi et al., 2012; Clement et al., 2019; Zhu et al., 2011) and restoring those levels to a youthful state is believed to have beneficial effects on health and longevity. 

Key Questions 

This analysis seeks to answer the following questions:

• Which health and/or longevity benefits result from raising NAD+ levels in humans? 

• Which risks are involved in raising NAD+ levels (general and method-specific)?

• What are the potential risk mitigation strategies?

• Which method or combination of methods is most effective in raising NAD+ levels?

• Which of the available methods are safe for use? 

• What is the best therapeutic protocol available at the moment?  

Impatient readers may choose to skip directly to  Section 6  for the conclusion and tips on practical application. 

Section 2: Methods

Analytic model

The RBA has been prepared based on the principles outlined in A Comprehensive Approach to Benefit-Risk Assessment in Drug Development ( Sarac et al., 2012 ). 

Literature search

A literature search was conducted on Pubmed and the Cochrane Library using the search terms shown in Table 1 . Titles and abstracts of the resulting studies were screened and relevant articles downloaded in full text. The references of the full-text articles were manually searched in order to identify additional trials that may have been missed by the search terms.

The inclusion criteria for human studies were necessarily broad as there is a scarcity of evidence. We chose to include any trial that was conducted in humans using NAD precursors other than niacin/nicotinic acid as the majority of those trials used cholesterol-lowering as the primary endpoint and don't mention NAD+ levels. Additionally, niacin/NA results in uncomfortable flushing in many people at relatively low doses and is therefore not generally used for NAD+ restoration therapy. 

Table 1: Literature Search 

Recommended Reading

The following sites offer information on NAD+ at a consumer level and are useful as an introduction to the topic:

•  

  • NAD Popular - alivebynature.com

  • An Interview with Mike Bonkowski – NAD+ and Aging - leafscience.org

  • NAD+ Repletion Shows Potential Against Aging - leafscience.org
    

  • NAD An Emerging Framework For Life Extension - anti-agingfirewalls.com

Abbreviation list

Section 3: Existing evidence

Summary of results from clinical trials (humans)

We identified 41 completed clinical trials and 29 that are ongoing ( aboutnad.com). The majority of published clinical trials have been performed for dermatological conditions and used NAM as the study compound.  Only two human studies used NAD+ as the study compound, one as i.v. therapy and the other topically. The other trials examined various precursors that have been shown to raise NAD+ levels. We identified a case report series but were unable to find any RCTs on systemic NAD+ i.v. or patch therapies. 

Table 2: Clinical Trials

Summary of results from preclinical trials (animals & in vitro )

Due to the lack of evidence from clinical trials, a decision was made to include results from preclinical studies (animals/in vitro) in order to gain more data to aid in the assessment of the risk/benefit ratio. We identified 145 preclinical trials with potentially clinically relevant risk/benefit criteria. The majority of the studies were conducted in rodents and studies on NAD+ itself as well as all known precursors exist.

Table 3: Preclinical trials 

Section 4: Risk-Benefit Analysis

Decision Model

Risk and benefit criteria

The decision profile is made of up risk and benefit criteria extracted from the outcomes of the above-mentioned papers. The benefit criteria are organized by category and include the type, magnitude, and duration of the benefit as well as its perceived importance to the patient. The risk criteria are organized by category, type, severity, frequency, detectability, and mitigation. All are assigned numerical values: 

1 = low

2 = moderate

3 = high

The numerical values for both risk and benefit criteria are then summarized serving as the justification for the weighting in the following column.

Weight

The criteria are weighted on a value scale to enable comparison (based on the relative importance of a difference). Each benefit and risk criteria is assigned a weight/importance of 1 (low) 2 (medium) or 3 (high).

Weighting is independent of data sets and the final weights are based on consensus with justification based on the preceding columns of the table.

Score

Each category is assessed according to the performance of NAD+ restoration therapy against the comparator (physiological aging) whereby a numerical value is assigned for each criterion -1 (inferior), 0 (equivalent or non-inferior) and +1 (superior) to the comparator.

Uncertainty

Uncertainty is determined according to the amount and quality of the evidence, whether it came from human or animal studies and whether methodological flaws, conflicting studies, or conflicts of interest (funding) by the authors are present. Human evidence is initially assigned a score of "1", evidence from rodent studies, "2", and in vitro or lower animal studies, "3". The uncertainty score is then adjusted by upgrading or downgrading using the above-mentioned criteria. 

Weighted score

The weights and scores are multiplied to produce weighted scores that enable direct comparison (-3 → +3) and then adjusted using the uncertainty score. Weighted scores may be upgraded where the uncertainty of the evidence is low or downgraded where the uncertainty of the evidence is high. 

Benefit assessment 

Table 4: Benefit assessment  

Benefits by system

Skin

Analysis of NAD+ in human skin samples has shown that there is a depletion in total NAD(H) pool content as well as an increase in PARP activity as a function of age ( Massudi et al., 2012 ). Thresholds for vitamin B3 deficiency vary by tissue, with the skin being highly susceptible as evidenced by its involvement in the symptom complex of pellagra. Evidence for the beneficial effect of NAD(H)-related compounds on the skin is relatively extensive dating back over 50 years. Many studies with relatively large sample sizes have been conducted in humans. 

Most trials examined the effect of NAM as a topical treatment although studies with oral NAM and topical NAD(H) treatment have also been performed. 

NAM applied topically and orally has been shown to decrease the immune suppression that occurs following UV exposure (Damian et al., 2008; Yiasemides et al., 2008, Sivapirabu et al., 2009). It is hypothesized that NAM works by preventing ATP depletion and glycolytic blockade (Park et al., 2010), thereby decreasing the level of immunosuppression induced by UV radiation and as well as through the enhancement of DNA repair. The downregulation of IL-10 may also play a role in NAM's anti-immunosuppressive effects (Monfrecola et al., 2013).

There is also high-quality evidence that oral NAM is effective in decreasing the rate of new nonmelanoma skin cancers (Chen et al. 2015; Drago et al., 2017; Surjana et al., 2012). The benefits were temporary, lasting only for the duration of treatment ( Chen et al. 2015) and stronger  effects were seen in higher-risk patients, paralleling the results of many NAD+ studies. Additionally, multiple studies have shown a decrease in the size of existing actinic keratoses and even complete regression in many patients (Chen et al., 2015; Drago et al., 2017). 

In photo-damaged skin, topical application of NAM and MN has been shown to increase stratum corneum thickness by approximately 70%, epidermal thickness by 20%, epidermal renewal 6-11%, as well as increasing the minimal erythemal dose, and decreasing transepidermal water loss (Jacobson et al., 2007; Tanno et al., 2000).

NAD-related compounds have also been used successfully in the treatment of several dermatological diseases:

• NAM has been used to treat acne (Shahmoradi et al., 2013; Shalita et al.,1995), melasma (Navarrete-Solıs et al., 2011), and pemphigus vulgaris (Iraji & Banan, 2010)

• MeNAM (the main metabolite of NAM) has been used in the treatment of rosacea (Wozniacka et al., 2005)

• NADH has been used in the treatment of rosacea and contact dermatitis (Wozniacka et al., 2003)

• NAD+ in the treatment of psoriasis (Wozniacka et al., 2006 ).

 The reported mechanisms of action behind the improvements are:

• increased synthesis of ceramides, free fatty acids, and cholesterol which are contained in the intercellular spaces of the horny layer ( Rolf, 2014 )

• sebo-suppressive, anti-inflammatory, and wound healing effects ( Rolf, 2014 )

• increased synthesis of collagen and proteins involved in the formation of keratin, filaggrin, and involucrin that improve the overall structure, moisture, and elasticity of the skin ( Rolf, 2014 )

• suppressing the transfer of melanosomes from melanocytes to keratinocytes ( Hakozaki et al., 2002 )

Preclinical

Oral niacin and topical NAM have been shown to reduce the incidence of skin cancer in mice and to prevent immunosuppression induced by UV radiation (Gensler et al., 1999; Gensler et al., 1997). An in vitro study of human keratinocytes found that NAM worked by preventing UV-induced ATP loss and glycolytic blockade rather than on ROS formation or apoptosis, suggesting that it exerts its effects via cellular energetic pathways (Park et al., 2010). NAM was found to protect dose-dependently as well as restore glycolytic rates concurrent with restoring ATP in oxidative stress (Rovito & Oblong, 2014). NAM has been shown to increase both the proportion of cells undergoing excision repair and the repair rate in each cell ( Surjana et al., 2013) and has antioxidant, immunomodulating and anti-inflammatory properties.   It has also proven useful in wound healing, increasing skin flap survival from 45% to 85% in rats (Collins et al., 1991). 

Cardiovascular system

Human studies on the benefits of NAD+ restoration for the heart and vessels are limited. A pharmacokinetic study of NR in humans recognized a trend towards decreased blood pressure and arterial stiffness though it did not reach significance (Martens et al., 2018) while a study that used NRPT as a precursor found a significant decrease in diastolic pressure at the lower dose tested, but not at the higher dose (Dellinger et al., 2017). A recent clinical trial that attempted to verify this trend found that NR supplementation had no effect on cardiometabolic parameters (Elhassan et al., 2019).

Two studies reported a significantly reduced maximum heart rate after treatment with NADH (Alegre et al., 2010; Castro-Marrero et al., 2016). A clinical study following cardiac surgery found that there was a decrease in cardiac injury markers with short term NAM administration (Mehr et al., 2018).

Rodent trials involving NAD+ restoration and the cardiovascular system are extensive. Several precursors have been shown to prevent cardiac remodeling and preserve cardiac function in heart failure models in mice (Cox et al., 2002; Pillai et al., 2010; Lee et al., 2016; Diguet et al., 2017; Martin et al., 2017; Zhang et al., 2017; Zhang et al., 2016). These effects are partially mediated by an increase in sodium channel conduction velocity due to a reduction in mitochondrial ROS (Liu et al., 2013).

NMN restored cardiac contractility, reduced cell death and decreased the expression of inflammatory markers in a model of pressure overload cardiomyopathy (Zhang et al., 2017). NMN has also been shown to reverse age-related arterial dysfunction (de Picciotto et al., 2016) and to extend the time rats could survive severe hemorrhagic shock by 25% through mitigation of inflammation, and improvements in cellular metabolism (Sims et al., 2018). 

Restoration of NAD+ levels  also has beneficial effects on infarct size (Yamamoto et al., 2014; Guan et al., 2016; Zhang et al., 2016). 

NAM has been shown to reduce the death of cardiomyocytes under both normoxic and hypoxic conditions and to promote mitochondrial protection (Tong et al., 2012). NAM or FK866 after I/R also exerted a positive effect on acute lung injury (Su et al., 2007; Wu et al., 2017). 

NADH has been shown to lower systolic blood pressure in rats (Bushehri et al., 1998). 

In vitro, exogenous NAD+ protected against hypoxia-reperfusion injury in both time and concentration-dependent manners by attenuating apoptosis through the restoration of SIRT-1 activity and subsequent inhibition of p53 (Liu et al., 2014). Limiting NAD+ consumption through the use of PARP-1 inhibitors also improved metabolism and ATP levels in rat cardiomyocytes (Gero et al., 2015).

Ex vivo, exogenous NAD+ reduced arrhythmic susceptibility in a mouse model of Brugada syndrome (Liu et al., 2009).

Skeletal muscle and mitochondria

Sarcopenia is one of the hallmarks of physical aging. It has been reported that NAD(H) levels decrease in advance of the onset of sarcopenia and that this decrease is accompanied by lowered mitochondrial oxidative phosphorylation (Pugh et al., 2013). NAD(H) is directly involved in mitochondrial oxidative phosphorylation as a co-enzyme.

Many studies have indicated that during aging, a significant reduction in NAD levels is observed in skeletal muscle ( Camacho-Pereira et al., 2016 ; Frederick et al., 2016 ; Gomes et al., 2013 ; Mouchiroud et al., 2013 ; Yaku et al., 2018 ; Yoshino et al., 2011 ; Zhang et al., 2016 ). Overexpression of Nampt in skeletal muscle has been shown to preserve ability in endurance exercise, which typically declines in aged mice ( Frederick et al., 2016 ) and in mice lacking Nampt, NR and NAM supplementation ameliorated functional deficits and restored muscle mass (Frederick et al., 2016).

Data from human trials is extremely limited. A couple of studies reported an increase in muscle strength, or fatigue index (Dellinger et al., 2017; Dolopikou et al., 2019). However, these benefits were limited to the older population (Dolopikou et al., 2019). However, another clinical trial reported no change in handgrip strength even in elderly individuals (Elhassan et al., 2019).

Three clinical studies of NADH for chronic fatigue syndrome reported a decrease in fatigue (Castro-Marrero., 2016; Santaella et al., 2004; Forsyth et al., 1999) and one found that NADH was effective and faster than conventional therapy (Santaella et al., 2004).  Myalgia was also seen to decrease in CFS patients (Santaella et al., 2004, Forsyth et al., 1999) as were postexertional malaise, muscle weakness and arthralgia (Forsyth et al., 1999). 

Several rodent trials have shown promising results related to improvements in mitochondrial function following NAD+ supplementation. 

NR and NAD+ improved muscle function and reduced muscle fiber degeneration in mouse and zebrafish models of muscular dystrophy through an improvement in mitochondrial function, and structural protein expression and reductions in PARP activity, inflammation, and fibrosis (Ryu et al., 2016; Goody et al., 2012). NR also improved disease progression in a mouse model of myopathy by inducing mitochondrial biogenesis, stimulating the mitochondrial unfolded protein response and preventing mitochondrial ultrastructure abnormalities (Khan et al., 2014).  In another mouse model of mitochondrial disease, administration of NR markedly improved the respiratory chain defect and with it, exercise intolerance (Cerruti et al., 2014). 

Preclinically it has been shown that aging reduces both the number of and NAD levels within muscle stem cells (Zhang et al., 2016). NR administration to aged mice restores NAD levels and the number of stem cells to the levels of those of young mice as well as improving their grip strength, running endurance, and preventing cellular senescence and mitochondrial dysfunction (Zhang et al., 2016).

Additionally, NAM has been shown to attenuate the aging process in skeletal muscle of rats (Pajk et al., 2017).

NR and PARP-1 inhibitors were shown in vitro to improve mitochondrial function by restoring mitochondrial membrane potential and oxidative activity by increased transcription of mitochondrial transcription factor A and mitochondrial DNA-encoded respiratory complexes (Felici et al., 2015). NMN supplementation has been shown to improve mitochondrial respiration in circadian mutant mice (Peek et al., 2013). A single dose of NMN increased hippocampal mitochondrial NAD+ levels for up to 24 hours, with a resultant effect of reducing ROS levels (Klimova et al., 2019).

Eye & ears

The incidence of retinal degenerative diseases, including glaucoma and age-related macular degeneration (AMD), is strongly associated with age. These diseases are caused by retinal cell death and the following irreversible degeneration of retinal cell axons. NAD+ decline has been observed in association with retinal dysfunction (Johnson et al., 2018) in that it causes metabolic and SIRT-3 dysfunction that is then followed by photoreceptor death  (Lin et al., 2016). Long-term supplementation of NMN improved age-related retinal dysfunction ( Mills et al., 2016 ) and also rescued vision in mice (Lin et al., 2016). 

Glaucoma is one of the most common causes of age-related blindness and is characterized by the progressive loss of retinal ganglion cells and their axons due to increased intraocular pressure. The relationship between NAD+ and axonal degeneration has been extensively investigated. It has been reported that oral administration of NAM reduces the risk of glaucoma 10-fold in mice through prevention of the decline of retinal NAD+ levels and subsequent mitochondrial dysfunction in retinal ganglion cells ( Williams et al., 2017).

AMD is the leading cause of irreversible vision loss in the elderly ( Ehrlich et al., 2008 ). It is associated with progressive degeneration of photoreceptors and retinal pigment epithelium (RPE) cells. Increased oxidative stress during aging is considered a prominent trigger of this process. It has been reported that exogenous administration of NAD+ suppresses oxidative stress-induced cell death in RPE cells ( Zhu et al., 2016) and appears to promote autophagy and reduce the production of ROS in RPE cells exposed to chronic oxidative stresses during aging (Zhu et al., 2016). NAM administration has also been shown to reduce the production of inflammatory cytokines and complement factors and inhibit the accumulation of drusen proteins, thus increasing RPE cell survival (Saini et al., 2017).

Presbycusis is an age-related impairment of hearing ability characterized by irreversible degeneration of cochlear hair cell and spiral ganglion neurons. Increased oxidative stress and mitochondrial DNA deletion during aging may play a major role in the pathophysiology. NR administration has been shown to rescue mice from noise-induced hearing loss whether administered before or after noise exposure via a SIRT-3 dependent mechanism ( Brown et al., 2014 ). Thus, it is hypothesized that administration of NAD+ precursors may prevent presbycusis in humans. 

Healthspan/regeneration/longevity

Data on the effect of NAD restoration on healthspan, regeneration, and longevity in humans is lacking. 

NMN and NR have been shown to prevent age-dependent declines of NAD+ in several studies in rodents (Gomes et al., 2013; Mills et al., 2016; Zhang et al., 2016). Long term administration (12 months) of NMN mitigated the age-associated physiological decline in mice by suppressing age-related body weight gain, enhancing energy metabolism, increasing physical activity, improving insulin sensitivity, and plasma lipid profile, and improving eye function. It prevented the gene expression changes in key metabolic organs that usually accompany aging and enhanced mitochondrial metabolism (Mills et al., 2016). 

Use of NAD+ precursors leads to multiple positive effects in terms of organ regeneration and stem cell maintenance in mice. Liver regeneration was increased (48% increase in mitotic activity) when NR was administered prior to partial hepatectomy (Mukherjee et al., 2017) and d ietary supplementation with NAM has been shown to restore DNA excision repair activity in mice exposed to radiation (Batra et al., 2014). 

In aged mice, NR restores colony formation, number of intestinal stem cells and rescues functional defects in the gut (Igarashi et al., 2016). The same study found that rapamycin blocked the rescue action of NR, raising questions about the effects of combining various rejuvenation therapies.  Adequate intracellular  NAD+  content is crucial for the maintenance of pluripotency in stem cells. In a study comparing NAD+, NA and  NAM, NAM  was found to be the sole agent capable of accelerating stem cell  proliferation and protecting stem cells from apoptosis and se nescence by reducing oxidative stress, reactive oxygen  species accumulation, and preventing mitochondrial membrane potential collapse (Son et al., 2013).

NAM and NR have been shown to prolong the worm lifespan by 16% (Mouchiroud et al., 2013). The worms also showed improvements in healthspan such as increased mobility in old age and increased mitochondrial function. The metabolite MeNAM has also been shown to prolong lifespan in worms as an independent factor (Schmeisser et al., 2013).  NR administration slightly extended the lifespan of aged mice (Zhang et al., 2016). No longevity study has been conducted using NMN but it did extend healthspan and lifespan in worm and mice models of ataxia-telangiectasia (Fang et al., 2016). One paper demonstrated that NAM supplementation improves aspects of healthspan but does not alter lifespan (Mitchell et al., 2018).

Metabolism

Aging causes many changes in metabolism including increased adiposity that accelerates aging by inducing low-grade inflammation in various tissues. Numerous studies have demonstrated that low NAD+ levels are associated with metabolic diseases such as diabetes, dyslipidemia, and non-alcoholic fatty liver disease (NAFLD) (Okabe et al., 2019;  Fang et al., 2017 ). 

Again, there is minimal data available in humans. One clinical trial found that 12 weeks of supplementation with NR had no effect on metabolic parameters (glucose tolerance, insulin resistance, lipolysis, etc.) in obese, insulin-dependent men (Dollerup et al., 2018). 

However, many preclinical studies have demonstrated that nutritional supplementation of NAD+ precursors ameliorates insulin resistance during obesity and aging (Canto et al., 2012; Gomes et al., 2013; Mills et al., 2016; Trammell et al., 2016; Uddin et al., 2016; Yoshino et al., 2011), most likely due to activation of the sirtuin pathway.

NR was found to induce a thermogenic response in lean mice that reduced visceral fat deposits, slightly increased oxygen consumption and increased body temperature (Crisol et al., 2018). NR has also been shown to inhibit weight gain in mice (Xie et al., 2019), to have favorable effects on triglyceride levels (Mardinoglu et al., 2017), and to prevent and revert NAFLD (by inducing a sirtuin (SIRT)1- and SIRT3-dependent mitochondrial unfolded protein response) (Gariani et al.,2016; Zhou et al., 2016). It also markedly reduced collagen synthesis (fibrosis) in models of hepatic steatosis (Pham et al., 2019) and exerted positive effects in alcohol-induced liver disease by lowering TG accumulation and elevating the hepatic NAD+ levels (Huang et al., 2018).

NMN has been shown to improve gene expression related to oxidative stress, the inflammatory response, and circadian rhythm, partially via SIRT-1 in diabetic mice (Yoshino et al., 2011) and to increase NAD levels in the liver, and improve hepatic insulin sensitivity and secretion in mice fed a high-fat (Ramsey et al., 2008) or fructose-rich diet (Caton et al., 2011). 

NADH has been shown to lower total cholesterol and LDL in hypertensive rats (Bushehri et al., 1998).

NA has also been shown to ameliorate chronic alcohol-induced fatty liver in rats (Li et al., 2014).

NAD+ supplementation significantly attenuated the weight gain in obese mice fed a high-fat diet (Roh et al., 2018) and recovered the suppressed rhythms in the diurnal locomotor activity pattern (Rho et al., 2018).

NAM improves glucose homeostasis in mice on a high-fat diet (Mitchell et al., 2018) and prevented diabetes by antagonizing an increase in NO and inhibiting B cell apoptosis (Alenzi, 2009). 

Nervous system

There is a gradual but significant decline in the NAD+/NADH ratio in the human nervous system during normal aging ( Zhu et al., 2015 ). This decline suggests a shift of the metabolic balance towards slower oxygen metabolism in the mitochondria, with a resultant lower ATP production rate.

Human studies on the benefits of NAD+ restoration in the nervous system are lacking. Up until now, all clinical trials have used oral or i.v. NADH. Positive results from these trials include reduced anxiety (Alegre et al., 2010), decreased sleep disturbances (Santaella et al., 2004; Forsyth et al., 1999), improvements in cognitive function (Birkmayer, 1996), fewer headaches (Forsyth et al., 1999), decreased symptoms of jet lag (Birkmayer et al., 2002), prevention of the progression of dementia, increased verbal fluency and visual-constructional ability (Demarin et al., 2004),  improvement of the motor symptoms of Parkinson's disease (Birkmayer, 1993), and increased bioavailability of levodopa in the plasma (Kuhn et al., 1996).

Studies in rodents suggest benefits in almost all areas of dysfunction in the nervous system. Maintaining/replenishing the NAD+ levels in the brain has been suggested to reduce ischemic damage and reperfusion injury after stroke ( Ying, 2007 , Zheng et al., 2012, Klaidman et al., 2003 ), protect against neuronal death ( Park et al., 2016 , Bi et al., 2012, Hou et al., 2018 ), promote remyelination ( Wang et al., 2017 ), and lessen spinal-cord reperfusion injury ( Xie et al., 2017) through reduction of the oxidative stress level and neuronal apoptosis (Xie et al., 2017).

Stroke

Supplementing NAM elevates brain NAD+ levels and substantially restores ATP levels via PARP and SIRT1 inhibition following stroke (Klaidman et al., 2003; Liu et al., 2009). These effects lead to a significant decrease in infarct size and improved neurological outcomes (Mokudai et al., 2000). NAM treated mice show higher levels of remyelination and less functional deficits than controls (Wang et al., 2017). However, in another study, NAM showed no effect on ischemic brain injury (Ying et al., 2007).

Intranasal administration of NAD+ has also been shown to profoundly decrease ischemic brain injury (Ying et al., 2007). Experiments show that the positive effects of NAD+ on infarct size, brain edema, and spinal cord I/R injury-induced apoptosis are at least partially related to blocking autophagy (Zheng et al., 2012; Xie et al., 2017b). 

The combination of NAD+ and NADPH was highly effective in protecting neurons against oxygen-glucose deprivation/reperfusion injury. It provided a larger therapeutic window and significantly increased the levels of ATP, improving long term mortality, and functional recovery (Huang et al., 2017).

NAD+ and NMN promote stem cell activation and neurogenesis after stroke, decrease infarct size and neurological deficits and enhance survival (Zhao et al., 2015).  A trial of NMN in a murine model of stroke showed that protection was highest at lower doses and that above doses of 62.5 mg/kg there was actually decreased survival of neurons (Park et al., 2016). NMN has also shown to prevent complications associated with tPA treatment such as brain damage, hemorrhage, neuroinflammation, and activation of MMPs (Wei et al., 2017).

In vitro, NAD+ and NAM are capable of inhibiting apoptotic neuronal death following glutamate toxicity and oxygen-glucose deprivation via preservation of mitochondrial biogenesis and integrity (Wang et al., 2014, Bi et al., 2012, Wang et al., 2008) and act to restore DNA repair activity Wang et al., 2008). Restoration of NAD+ levels in mouse astrocytes enable recovery of mitochondrial membrane potential and prevent cell death (Alano et al., 2004). 

Neurodegeneration

NAM has been found to preserve mitochondrial integrity, autophagy function and to reduce AB and p-tau pathologies, resulting in improved cognitive performance (Liu et al., 2013). Another study identified that NAM reduced a specific species of tau, and increased microtubule stability through a mechanism involving sirtuin inhibition (Green et al., 2008). 

It has been reported that NR administration to AD model mice ameliorates cognitive function ( Gong et al., 2013, Hou et al., 2018 ), contextual memory ( Sorrentino et al., 2017 ), short term spatial memory, contextual fear memory (Xie et al., 2019), hippocampal synaptic plasticity (Hou et al., 2018), reduces the levels of Aβ plaques (Gong et al., 2013; Xie et al., 2019), reduces phospho-tau pathology (Xie et al., 2019), reduces DNA damage, neuroinflammation and apoptosis,  and increases the activity of SIRT-3 in the brain (Hou et al., 2018). 

Trials with NMN have  shown improvements in cognitive impairment, sensory processing (Johnson et al., 2018), mitochondrial function (Long et al., 2015), AB pathology, angiogenic capacity (Kiss et al., 2019), vasodilation (Tarantini et al., 2019) and neuroinflammatory parameters (TNF, IL-1, IL-6) (Yao et al., 2017). It was found to reduce SIRT1 activity, preserving NAD+ for energetic metabolism (Long et al., 2015).

NADH was found to improve learning abilities in aged rats (Rex et al., 2004). 

Other nervous system diseases

NA administration has been shown to reverse Schwann cell maturation defects and in doing so, to prevent the development of peripheral neuropathy (Sasaki et al., 2018).

NR has also been shown to prevent the development of diabetic (Trammell et al., 2016) and paclitaxel-induced peripheral neuropathies (Hamity et al., 2017). 

In Drosophila models of Parkinson's disease, NAM was found to improve mitochondrial function and have neuroprotective effects (Lehmann et al., 2016) as well as improve the motor deficits (Jia et al., 2008). NAD+ supplementation prevented dopaminergic neurodegeneration and deficits in behavior (Caito & Aschner, 2016) in worm models. 

NAM has been shown to reduce acute cortical neuron death and edema in the traumatically injured brain in rats (Hoane et al., 2006). 

Enhancement of the NAD+ salvage pathway was found to revert the toxicity of primary astrocytes with ALS linked mutant SOD1 toward co-cultured motor neurons (Harlan et al., 2016).

Reproduction

Human trials on this topic are nonexistent. A recent study in mice that examined the effect of supplementing nursing mothers with NR found benefits for the mother (postpartum weight loss) and for the offspring (physical performance, anti-anxiety, spatial memory, delated onset of behavioral immobility) (Ear et al., 2019). Supplementation with NA has been shown to prevent congenital malformations (Shi et al., 2017) and improve the quality of aged oocytes (Wu et al., 2019). 

Immune

Supplementation with NR in humans led to decreased levels of circulating inflammatory cytokines including IL- 2,5,6 and TNF-a (Elhassan et al., 2019). 

Trials on NADH in chronic fatigue syndrome reported a decrease in lymphadenopathy (Santaella et al., 2004; Forsyth et al., 1999) and allergies (Forsyth et al., 1999). NADH has also been shown to increase lymphocyte proliferation in vitro (Bouamama et al., 2017).

NAM increased oxidative burst activity in patients with Type 2 diabetes (Osar et al., 2004) and has been found to decrease TNF-a secretion, likely through SIRT inhibition, thus protecting mice from endotoxic shock (Van Gool et al., 2009). NMN has been shown to reduce lactic acidosis and IL-6 in hemorrhagic shock which are strong predictors of mortality (Sims et al., 2018). 

In vitro , NAM administration was protective of DNA damage-induced death of mononuclear cells (Weidele et al., 2010). NR, MeNAM, and NAM have all been shown to decrease cancer cell survival in a leukemic cell line (Petin et al., 2019). 

NADH has been shown to decrease oxidative stress in aged lymphocytes (Bouamama et al., 2017).

Kidney

Renal levels of NAD+ and SIRT-1 activity decline with age (in mice) (Guan et al., 2017). 

A clinical study on NAM use in chronic renal disease showed a significant increase in HDL and decrease in LDL but no effect on triglycerides, in addition to its main purpose of controlling hyperphosphatemia (Takahashi et al., 2004). A second study evaluated the use of NAM on the incidence of acute kidney injury (AKI) following cardiac surgery and identified a significant decrease in AKI events as well as decreased cardiac injury markers (Mehr et al., 2018). 

Mice that were nephrectomized to mimic chronic kidney disease and supplemented with niacin showed improvements in hypertension, proteinuria, glomerulosclerosis, and tubulointerstitial injury (Cho et al., 2009). There was also a trend toward lowering serum creatinine and raised creatinine clearance. In models of AKI caused by cisplatin, mice were protected by the administration of NMN (Guan et al., 2017). Another murine study found that NAM over 4 days was able to reverse established AKI (Tran et al., 2016). NAM has been shown to inhibit renal interstitial fibrosis in mice (Zheng et al., 2018) through the suppression of apoptosis, T cell, and macrophage infiltrations and induction of proinflammatory cytokines and fibrotic proteins (Zheng et al., 2018).

Gene expression

Supplementation with various NAD+ precursors has also been shown to have potentially beneficial effects on gene expression. In humans, supplementation with NR increased gene expression related to cell adhesion, actin cytoskeleton organization, and cell motility (Elhassan et al., 2019). In several mammalian studies, NR and NMN have been shown to increase SIRT 1 and SIRT 3 activity (Li et al., 2015; Pajk et al., 2017; Cantó et al., 2012; Hou et al., 2018). In mice, NMN has also been shown to increase gene expression of genes that lead to an improvement in oxidative stress status, inflammatory response and circadian rhythm (Yoshino et al., 2011).

Risk assessment 

Table 5: Risk assessment  

Individual risks

Acute adverse drug effects

The acute adverse drug effects in humans include thrombocytopenia, diarrhea, nausea, skin rash, flushing, leg cramps, erythema, pruritis, burning skin, fatigue, abdominal discomfort, and headache (see Table 2). The most common side effects are GI upsets. High doses of NAM caused nausea and vomiting in a pharmacokinetic study (Dragovic et al., 1995). However, all side effects are minor, relatively infrequent, reversible and easily detectable. 

Tumorigenesis, invasion, and motility

One major concern with increasing NAD+ levels is whether it leads to an increased risk of cancer and/or faster growth of tumors. To date, there is no data from clinical trials addressing this risk. However, in support of this view, NAD+ depletion has been explored as a means of controlling/decreasing tumor growth and these studies have shown positive results ( Takao et al., 2018 ,  van Horssen et al., 2012, Ginet et al., 2014) while restoration of NAD+ levels by supplementation has abolished the positive effects. 

An increase in NAD(H) pool size and NAD+: NADH ratio due to activation of the salvage pathway was identified in human tissue samples of colorectal cancer and mice ( Hong et al., 2019 ). The increases in NAD(H) inhibit the accumulation of ROS, allowing the tumor to grow. In an in vitro study, pharmacological and genetic inhibition of NAMPT decreased NAD+ levels and glioblastoma-like stem cell self-renewal capacity, and NAMPT knockdown inhibited the in vivo tumorigenicity of GSCs (Gujar et al., 2016). A recent preclinical trial  has shown that NAD metabolism clearly promotes the progression of pancreatic cancer through enhancement of the inflammatory environment (Nacarelli et al., 2019).

On the other hand, increasing NAD+ levels locally, in the skin, has been shown to markedly reduce the progression of precancerous skin lesions to squamous cell carcinomas ( Jacobson et al., 2007 ) likely via increased DNA repair activity, driven by the increased NAD+ that is available for use by reparative enzymes. NAD+ precursor supplementation has also been shown to prevent and reduce the size of established hepatic tumors (Tummala et al., 2014), decrease the survival of tumor cells (Petin et al., 2019) by increasing oxidative stress (Zhao et al., 2011) and increasing autophagy (Han et al., 2011).

In summary, increasing NAD+ levels may be tumorigenic or accelerate the growth of existing lesions under stressed conditions such as in premalignant senescent lesions.

Buildup of metabolites

Only a few studies have measured the effects of increasing NAD+ levels on the complete NAD metabolome ( Trammell et al., 2016, Elhassan et al., 2019 ). Most metabolites are elevated in response to precursor treatment ( Trammell et al., 2016 ) and a better understanding of the potential consequences of increased levels of the various metabolites is necessary.

For example, NR is a direct precursor of NAAD and has been shown to generate high levels of NAAD in the murine liver and heart (45-fold higher levels following a single dose compared to an NAD+ rise of 2.3 fold) (Trammell et al., 2016). In humans, NAAD levels in muscle and blood rose 2-fold and 4.5 fold respectively with NR supplementation while NAD+ levels were unchanged in muscle and rose 2 fold in the blood (Elhassan et al., 2019). NAAD normally circulates at very low levels. Its physiological role other than being an NAD+ precursor is unclear though once phosphorylated to NAADP it is involved in Ca signaling.

Some preclinical studies suggest that high levels of NAM, NMN, and MeNAM may also cause adverse effects (Liu et al., 2018; Parsons et al., 2003;  Naia et al.,2017;  Mori et al., 2012). One dose of NR also resulted in an 8.4-fold higher plasma level of 2PY, the methylated metabolite of NAM (Trammell et al., 2016). Clinical trials of NR at doses of 1000 mg/day also showed increases between 5 and 10 fold in MeNAM and 2-PY concentrations (Conze et al., 2019; Elhassan et al., 2019).

It has been suggested that at high levels, NAM can act as a uremic toxin contributing to thrombocytopenia ( Rutkowski et al., 2003 ,  Lenglet et al., 2016 ). This is supported by reports of thrombocytopenia and increased bruising in some clinical trials (Airhart et al., 2017;  Martens et al., 2018). Trials on NAM supplementation in kidney disease reported extremely high levels of 2-PY with resultant thrombocytopenia as well as increased MeNAM and NMN levels (Mehr et al., 2018). It has been proposed that this results from a NAM-induced drop in the serum level of thyroxin-binding globulin or one of its derivatives ( Lenglet et al., 2016 ). 

In vitro, mouse neuronal cell survival rate dropped when the cells were cultured with MeNAM or NAM (Mori et al., 2012). NAM and MeNAM are able to cross the blood-brain barrier and at high concentrations can have a toxic effect on neurons, as shown in Parkinson’s and Huntington’s disease models ( Parsons et al., 2003;  Naia et al.,2017;  Mori et al., 2012; Harrison et al., 2018).

Epidemiological surveys suggest that niacin may play a role in Parkinson’s disease, in that niacin deficiency appears to protect against it. The average NAM intake in western populations is around 35 mg a day while the recommended daily allowance is 15 mg a day. Assuming a 20 mg/day overdose, that equates to 350 g over 50 years. Even over an extended period, that is a large number of toxin equivalents reaching neurons.

It has been suggested that superoxides formed by MeNAM via complex I destroy complex I subunits either directly or indirectly via mitochondrial DNA damage (Fukushima, 2005). NAM exacerbated dopaminergic degeneration, behavioral deficits and structural brain changes in rats (Harrison et al., 2018). High concentrations of NAM also blocked mitochondrial-related transcription in an in vitro Huntington's disease model, worsening the motor phenotype (Naia et al., 2017).

Exposure to MeNAM also markedly reduced liver NAD content and NAD/NADH ratio, while increasing H202 production and insulin resistance, leading to the hypothesis that NAM overdose may play a role in the development of diabetes. In human diabetics, MeNAM levels were significantly higher after an oral dose of NAM than in controls (Zhou et al., 2009).  On the other hand, MeNAM supplementation has been shown to extend the lifespan of worms (Schmeisser et al., 2013). 

In vitro administration of NR and NMN showed no effect on ATP levels and increased NAM 9-fold and 1.8-fold respectively (Oakey et al., 2018). NAM, as a SIRT inhibitor, has been shown to negatively affect lifespan in yeast (Bitterman et al., 2002). NAM treated rats also had higher hepatic and renal markers of DNA damage, and impaired glucose sensitivity and glucose tolerance (Li et al., 2009). NAM supplementation can lead to decreased methylation stores (Li et al., 2009) and has been shown to disturb monoamine transmitter metabolism (Tian et. al., 2013).

In humans, a 100 mg dose of NAM induced an increase in MeNAM, norepinephrine, and homocysteine and a decrease in metanephrine and betaine leading the authors to conclude that high NAM intake may be involved in cardiovascular disease (Sun et al., 2012). In contrast, a recent study found that homocysteine levels are not increased by NR supplementation (Conze et al., 2019) suggesting it is a precursor specific effect. 

Cumulative doses of NA also increase MeNAM and H202 levels and are associated with a decrease in liver and skeletal muscle glycogen levels. This increase may lead to oxidative stress, methyl group depletion, and insulin resistance, increasing the risk of diabetes (Li et al., 2012). 

Accumulation of NMN promotes axonal degeneration in cases of physical injury as well as in chemotherapeutic-induced mouse models of peripheral neuropathy (Di Stefano et al., 2015; Di Stefano et al., 2017). In one experiment, NMN levels rose 2.5 fold (≈ 4 nmol/g) after axonal injury (Di Stefano et al., 2015). Although the rise is due to the inability of the required enzyme to reach its place of action, exogenous delivery of NMN also promoted axonal degeneration. Additionally, NR is converted to NMN so if the enzyme (NNMAT) is overwhelmed or not functioning well, it could lead to an accumulation of NMN and axonal toxicity. Bypassing NMN production has been shown to protect neurons from chemotherapy-induced degeneration (Liu et al., 2018).

Therefore, it is important to consider that creating a state of NAD+ excess and increased levels of metabolites could have unintended effects in the CNS or elsewhere. This underscores the importance of monitoring metabolite levels during NAD+ therapy. 

Detrimental effects on exercise performance

A preclinical study ( Kourtzidis et al., 2016 ) found that exercise performance in young rats decreased by 35% after acute administration of NR. The rats were 4 months old, corresponding to a human age of 20 years and were given a high dose (300 mg/kg body weight = 48.6 mg/kg HED). Possible mechanisms for this decrease were explored in a follow-up study ( Kourtzidis et al., 2018 ) and it was found that NR supplementation exerted several effects on redox-related markers (increased NADPH and glycogen in the liver, increased F2‐isoprostanes in plasma, decreased glutathione peroxidase, glutathione reductase, and
catalase in erythrocytes, and decreased glucose and maximal lactate accumulation in plasma). These findings support the hypothesis that exogenously administered redox agents in healthy populations might lead to adverse effects.

A follow-up study was performed in humans ( Dolopikou et al., 2019 ) that compared the effects of acute NR administration on redox status and exercise performance in young (22.9 years old) and old (71.5 years old) age groups. It was found that although NR raised NAD(P)H levels in both groups, redox homeostasis, and exercise performance were improved only in the older group. This is in line with other studies that show a trend towards increased effectiveness of NAD supplementation in groups with the highest level of baseline dysfunction ( Martens et al., 2018 ). These results emphasize the importance of developing a set of age-related reference NAD values to aid in decision making about the appropriate time to commence NAD+ treatment. 

Inflammatory arthritis

Increasing NAD+ levels may also have negative effects on inflammatory conditions such as rheumatoid arthritis due to increased NAMPT activity and NAD+ use by immune cells ( Busso et al., 2008) . A murine study showed upregulation and worsening of arthritis in response to increased NAD+ and decreased arthritic severity and cytokine release when NAMPT was blocked and NAD+ levels were lower (Busso et al., 2008). There is no data in humans on this topic.

Negative effects on senescent cells

Senescent cells exhibit both protective (inhibition of tumorigenesis) and deleterious (accelerated aging, increased tumorigenesis) effects through a complicated sequence of activities that are beneficial at first but become proinflammatory later (Nacarelli et al., 2019). Senescent cell phenotypes vary according to cell type and the way in which senescence is induced (Mendelsohn and Larrick., 2019).

The NAD+/NADH ratio and NAD+ levels are significantly increased in oncogene-induced senescent (OIS) cells (Nacarelli et al., 2019) whereas the mitochondria dysfunction-associated senescence (MiDAS) secretory phenotype is linked to a lower NAD+/NADH ratio. A low proinflammatory SASP also accompanies replicative senescence (RS) (Nacarelli et al., 2019). 

It has recently been shown that exogenous NMN supplementation increases the strength of the proinflammatory SASP in OIS cells and converts a low proinflammatory SASP during RS into a high proinflammatory SASP (Nacarelli et al., 2019). Senescent cells with a high proinflammatory SASP have also been shown to induce senescence in neighboring cells (Mendelsohn and Larrick., 2019). NAD+ supplementation may result in an increased number of senescent cells with the detrimental high proinflammatory SASP. 

Feedback suppression

Interestingly, NAD+ levels appear to be downregulated chronically, though the level at which this occurs is unknown. This effect was observed in a clinical trial of NRPT in which a 40% elevation of NAD+ levels was sustained but a 90% elevation was not, despite continued treatment (Dellinger et al., 2017). Interestingly, the high dose treatment arm didn't experience as many beneficial effects as the moderate dose treatment arm. This downregulation raises the question of whether higher NAD+ levels eventually cause negative impacts on cell function, leading to an adaptive response. One theory is that the NAD+/NADH ratio is not controlled by the compounds but rather enzymatically, through NQ01 ( anti-agingfirewalls.com ). In support of this hypothesis, oral supplementation of NR has been shown to downregulate gene sets related to energy metabolism (Elhassan et al., 2019). 

Metabolism/Biochemistry

One human study of NRPT has shown a slight increase in LDL (Dellinger et al., 2017). However, another study on NR in overweight adults showed no significant difference, suggesting the elevation was related to pterostilbene and not NR.

One study also reported significant decreases in potassium levels, hematocrit, hemoglobin, and platelet count although none of these had any clinical significance. 

In rats, NAM administration at a dose of 500 mg/kg/day was found to increase lipid peroxidation in the liver (Melo et al., 2000). Several studies have found no change in blood levels of glucose, insulin, TG or HDL (Bushehri et al., 1998; Dollerup et al., 2018; Conze et al., 2019). NMN has been shown to slightly impair glucose tolerance in mice (Ramsey et al., 2008). 

Immune cells

It has been demonstrated that extracellular NAD+ induces apoptosis in naive T-cells (Liu et al., 2001) and that injection of NAD+ in mice can lead to concentrations capable of inducing sequestration and apoptosis of T cells in the liver (Liu et al., 2001). The steady-state concentration of NAD+ in the serum is 0.1 uM and the NAD+ that is released from lysed cells, exceeding this base level during inflammation, could limit the destructive action of autoreactive T cells (Liu et al., 2001). However, in the case of NAD+ supplementation, this could lead to a depression of the T-cell count. 

Summary of the evidence on form, dose, duration, and pharmacokinetics

NAM

The reported efficiency of NAD+ elevation after oral NAM supplementation (500-1000 mg/day) varies from a 30% increase (Yiasemides et al., 2008) to nearly 5 times baseline levels (Kjellen et al., 1986) while systemic absorption of topical NAM is reported to be approximately 10% (Cosmetic Ingredient Review (CIR) Expert Panel, 2005). 

A phase 3 randomized trial of NAM used 1000 mg/day for 12 months without any serious adverse effects (Chen et al., 2015). A second trial used 750 mg/day for 6 months and reported only one case of diarrhea as a side effect (Drago et al., 2017). NAM is generally well tolerated at doses of up to 6g per day for short term use (Dragovic et al.,1995) and has been used at 3 g/day over 3 years with minimal side effects (Knip et al., 2000). NAM has not shown any oncogenic effects in humans despite being used at high doses in more than 2000 people for several years (Knip et al., 2000). 

Topical NAM (0.3-5%) has been used for up to 12 weeks with no reported side effects (Bisset et al., 2006). One topical NAM trial reported local side effects of flushing and burning at a dose of 4% NAM (Iraji & Banan, 2010).

MN

Topical MN (1-5%, 0.5mL per day for 12 weeks) increased NAD+ levels in the skin by 25% with no reported adverse events (Jacobson et al., 2007). 

NMN

There are no published studies in humans. In mice, after injection or oral ingestion, NMN rapidly appears in plasma, liver, white adipose tissue, and pancreas leading to increased NAD+ (2~3-fold) concentrations in liver (Yoshino et al., 2011; Mills et al., 2016). It is still unclear whether NMN can cross the blood-brain-barrier, however, intraperitoneal administration rapidly increases NAD+ levels in the brain (Stein and Imai, 2014; Yoon et al., 2014). 

NR

Oral NR (100-1000 mg) has been shown to dose-dependently increase systemic NAD+ levels from 22% up to 2-3 fold (Mendelsohn and Larrick, 2017; Airhart et al., 2017; Dellinger et al., 2017; Martens et al.,2018; Trammell et al., 2016; Conze et al., 2019). The elevation was maintained until the end of an 8-week trial (Conze et al., 2019).

The highest dose of NR tested in humans in a published study is 2000 mg/day for a duration of 12 weeks ( Dollerup et al., 2018 ). Reported adverse events at this dosage were minor and included  pruritus, excessive sweating, bloating, and  transient changes in the stool. Following a dose of either 300 or 1000 mg NR, blood NAD+ levels in PBMCs peak at 8 hours (Conze et al., 2019).

Another study used 500 mg/day for 2 days with no reported adverse effects and reported that NADH levels increased about 50-60% and NADPH increased 32-38% (Dolopikou et al., 2019).

The current dose recommendation for NR is between 150-300 mg/day (3 mg/kg/day) based on a toxicology study where the lowest observed adverse effect level (LOAEL) for NR was 1000 mg/kg/day, and the no observed adverse effect level (NOAEL) was 300 mg/kg/day. That this dose is safe and efficient has recently been confirmed in a large clinical study (Conze et al., 2019) with the additional establishment of 1000 mg/day as the tolerable upper limit.

NRPT

One study (Dellinger et al., 2017) has shown that NR 250 mg/day combined with pterostilbene 50mg/day increased NAD+ levels by 40% at 30 days, while NR 500 mg/day combined with pterostilbene 100 mg increased NAD+ levels by 90%. However, by 60 days the levels slipped back to 55%. At these doses, a total of 48 minor adverse events were reported. In the same study, beneficial effects on liver enzymes were limited to the lower dose.

NAD+

To date, there is only one paper published that used i.v. NAD+ . This article dates from 1961 (O'Hollaren, 1961) and is a retrospective case series on the use of NAD+ in the treatment of addiction. It suggests a dose of 500-1000 mg added to 300 cc. of normal saline, at a rate between 5-35 drops per minute according to tolerance. In the treatment of addiction, it is to be given daily for 4 days, then twice per week for a month and finally as a maintenance dose twice per month until the addiction has been overcome. The author reported that no toxic effects occurred in over 100 patients treated in this manner. However, if administered too quickly patients complained of headache and shortness of breath (O'Hollaren, 1961). 

We identified one other document about the use of NAD+ as i.v. therapy (also for the treatment of addiction) that  states that NAD+ i.v. therapy is safe at 2 g/day or less. They suggest the use of 800-1800 mg/day of NAD+ by i.v. over a period of 3-8 hours for a duration of 7-16 days (Humiston, 2017).

NAD+ has also been used topically as an antipsoriatic (Wozniacka et al., 2006).

In vitro, intracellular NAD+ increased upon exposure of cell lines to exogenous NAD+ whereas NAD+ precursors could not reproduce the effects of exogenous NAD+ and were not found in the medium, suggesting direct cellular uptake. In mitochondria exposed to exogenous NAD+, NAD+, NADH and O2 consumption and ATP production were increased while DNA repair was unaltered (Pittelli et al., 2011).

NADH

Human trials that used between 5-30 mg oral NADH per day for up to two months reported no adverse effects (Alegre et al., 2010; Castro-Marrero et al., 2016; Mero et al., 2008; Birkmayer et al., 2002; Demarin et al., 2004 ). The longest study of NADH was 2 years at a dose of 5-10 mg/day with no reported adverse effects (Santaella et al., 2004). Another study that used 10 mg for 4 weeks reported only mild GI symptoms as a side effect (Forsyth et al., 1999).

One clinical trial used NADH 10 mg over 30min. as i.v. therapy for 7 days and also reported that there were no adverse events (Kuhn et al., 1996).

Acipimox

A trial on muscle mitochondrial function used 750 mg for 2 weeks with no reported side effects (van de Weijer et al., 2015).

Dose

The elevation in NAD+ levels achieved initially at higher dosages may not be maintained (Dellinger et al., 2017) nor is it necessarily more beneficial. In a trial of oral NAM, no greater efficacy was found in reducing immunosuppression with 1500 mg than with 500 mg daily (Yiasemides et al., 2008). Dose-response studies in mice have also indicated that lower doses of NMN may be more beneficial for neuronal outcomes (Mills et al., 2016; Park et al., 2016) as well as in decreasing liver enzymes and blood pressure (Dellinger et al., 2017). 

Pharmacokinetics

The pharmacokinetic profiles of NAD precursors show significant variation (see Table6 ). The half-life of NAD itself varies between 15 min and 15 h depending on the tissue. Some precursors preferentially affect certain tissues resulting in differential downstream effects ( Kirkland et al., 2009 ). As well, precursors may compete with each other in various reactions. For example, it has been proposed that NADH competes with NAD+ for binding to sirtuins and inhibits the catalytic activity of sirtuins ( Houtcooper et al., 2009 ). NAM can also noncompetitively bind to the sirtuins and acts as a very potent inhibitor of sirtuin activity ( Bitterman et al. 2002; Guan et al., 2014). Besides NAM and NA, only the corresponding nucleosides readily enter the cells. NAD and NMN undergo extracellular degradation resulting in the formation of permeable precursors. Precursors are converted to NMN in the cytosol for uptake into the mitochondria (Nikiforov et al., 2011). NR and NMN do not cause flushing ( Cantó et al., 2012). Only NR and NA increased NAD+ levels in muscle (Cantó et al., 2012). All precursors increased NAD+ levels in the liver (Cantó et al., 2012). NR increased NAD+ in liver and muscle but not in brain or white adipose tissue (Cantó et al., 2012). NADH and NAM levels were largely diminished in NR-fed muscles (Cantó et al., 2012). NMN must be converted to NR for uptake by some cell types (Cantó et al., 2012). 

Table 6: NAD precursors 

Section 5: Presentation of Results 

The following "tornado" diagram summarizes the results of the previous sections:

• The risk-benefit criteria are listed in the category column.

• The weighted score after factoring in uncertainty is shown as a numerical value.

• The weight of the criteria is proportional to the width of the columns. 

• Risk and benefit criteria are assigned to either low (1-1.66), medium (1.67-2.33), or high (2.34-3) weighted categories based on the results of the assessment in Table 4 and Table 5.

• The diagram is filterable by category so the main risks and benefits for each system can be viewed. 
  

To view the tornado diagram as a pdf please click on the thumbnail below:

Open

For those who would prefer to view the document in excel, we have included the original .xls file.

NAD Restoration Therapy

Section 6: Conclusions of the Risk-Benefit Analysis 

Main benefits

Restoration of NAD+ levels has been shown to have beneficial effects on several organ systems and diseases (Table 4) with an excellent acute toxicity profile. The main benefits are in diseases or conditions that threaten the energetic status of the cell such as ischemic stroke, heart failure/infarction, and mitochondrial diseases. The highest level of evidence for NAD+ restoration therapy in humans is for skin diseases. There is a multitude of potential benefits for which the evidence level is still quite low because of the lack of clinical trials.

Main risks

The major risks are related to tumorigenesis, the buildup of metabolites with undesirable effects, and an increase in the proinflammatory SASP of senescent cells (Table 5). These have not appeared in clinical trials to date but have been identified during mammalian preclinical trials. More clinical trials are necessary to adequately assess the risk of long term NAD+ supplementation.  Short and medium-term supplementation (up to twelve weeks) with NR elevates NAD+ levels safely and effectively but t here is a lack of studies examining the potential adverse health effects of chronic, year-long NAD+ supplementation.

Risk Mitigation Strategies

• Consult your physician before beginning therapy

• Assess baseline NAD+ metabolome values with a comparison to reference values from clinical papers to determine the appropriateness of beginning therapy.

• Repeat screening of the NAD+ metabolome at regular intervals to determine the effectiveness of the therapy, the appropriateness of the dose as well as to identify the accumulation of possible toxic levels of metabolites

• Screen for cancer before beginning therapy

• Avoid NAD+ restoration if cancer has been diagnosed or treated within the previous 5 years

• Assess senescent cell burden and remove senescent cells when possible before beginning NAD+ therapy

• Measure blood values, liver & kidney function and electrolyte values at regular intervals

• Self-monitor for signs of decreased exercise performance or fatigue

• Cease therapy if any identifiable adverse effects occur

• Exercise caution when combining NAD+ with other treatments

• Limit the duration of NAD+ restoration therapy as safety studies have only been performed up to 12 weeks in humans

Section 7: Practical Application

Form & Dose

• Clinical studies have shown that NAD+ levels are raised effectively by NR and NAM.

• The effectiveness and safety of the direct use of NAD+ by i.v. and patches in humans is unknown and therefore, cannot be recommended at this time.

• The same applies to NMN.

• NAM has the best safety profile according to the current evidence base. However, its role as a SIRT-1 inhibitor makes it questionable for use as a rejuvenation treatment.

• NADH has a good safety profile but there is a lack of data on its effectiveness in raising NAD+ levels.

• NR is currently the method of choice for raising NAD+ levels due to its proven effectiveness and safety profile.

• More isn't necessarily better. 

• The dose recommendation for NR is 150-300 mg/day (3 mg/kg/day).

• According to the latest evidence, 1000 mg/day has been established as the tolerable upper limit of NR for medium durations (up to 8 weeks).